ABSTRACT
The cyclin C-Cdk8 kinase has been identified as both a tumor suppressor and an oncogene depending on the cell type. The genomic locus encoding cyclin C (Ccnc) is often deleted in aggressive anaplastic thyroid tumors. To test for a potential tumor suppressor role for cyclin C, Ccnc alone, or Ccnc in combination with a previously described thyroid tumor suppressor Pten, was deleted late in thyroid development. Although mice harboring individual Pten or Ccnc deletions exhibited modest thyroid hyperplasia, the double mutant demonstrated dramatic thyroid expansion resulting in animal death by 22â weeks. Further analysis revealed that Ccncthyr-/- tissues exhibited a reduction in signal transducer and activator of transcription 3 (Stat3) phosphorylation at Ser727. Further analysis uncovered a post-transcriptional requirement of both Pten and cyclin C in maintaining the levels of the p21 and p53 tumor suppressors (also known as CDKN1A and TP53, respectively) in thyroid tissue. In conclusion, these data reveal the first tumor suppressor role for cyclin C in a solid tumor model. In addition, this study uncovers new synergistic activities of Pten and cyclin C to promote quiescence through maintenance of p21 and p53.
Subject(s)
Cyclin C/metabolism , PTEN Phosphohydrolase/metabolism , Thyroid Neoplasms/metabolism , Animals , Cell Line, Tumor , Cyclin C/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Mice , Mice, Knockout , PTEN Phosphohydrolase/genetics , STAT3 Transcription Factor/genetics , STAT3 Transcription Factor/metabolism , Thyroid Neoplasms/genetics , Thyroid Neoplasms/pathologyABSTRACT
Electrophilic carbonyl compounds are highly cytotoxic and genotoxic. Aldo-keto reductase 1B10 (AKR1B10) is an enzyme catalyzing reduction of carbonyl compounds to less toxic alcoholic forms. This study presents novel evidence that AKR1B10 protects colon cells from DNA damage induced by electrophilic carbonyl compounds. AKR1B10 is specifically expressed in epithelial cells of the human colon, but this study found that AKR1B10 expression was lost or markedly diminished in colorectal cancer, precancerous tissues, and a notable portion of normal adjacent tissues (NAT). SiRNA-mediated silencing of AKR1B10 in colon cancer cells HCT-8 enhanced cytotoxicity of acrolein and HNE, whereas ectopic expression of AKR1B10 in colon cancer cells RKO prevented the host cells against carbonyl cytotoxicity. Furthermore, siRNA-mediated AKR1B10 silencing led to DNA breaks and activation of γ-H2AX protein, a marker of DNA double strand breaks, particularly in the exposure of HNE (10 µM). In the AKR1B10 silenced HCT-8 cells, hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutant frequency increased by 26.8 times at basal level and by 33.5 times in the presence of 10 µM HNE when compared to vector control cells. In these cells, the cyclic acrolein-deoxyguanosine adducts levels were increased by over 10 times. These findings were confirmed by pharmacological inhibition of AKR1B10 activity by Epalrestat. Taken together, these data suggest that AKR1B10 is a critical protein that protects host cells from DNA damage induced by electrophilic carbonyl compounds. AKR1B10 deficiency in the colon may be an important pathogenic factor in disease progression and carcinogenesis. © 2016 Wiley Periodicals, Inc.
Subject(s)
Acrolein/toxicity , Aldehyde Reductase/metabolism , Aldehydes/toxicity , Colorectal Neoplasms/chemically induced , Colorectal Neoplasms/metabolism , DNA Damage/drug effects , Mutagens/toxicity , Acrolein/metabolism , Aldehyde Reductase/analysis , Aldehyde Reductase/genetics , Aldehydes/metabolism , Aldo-Keto Reductases , Cell Line, Tumor , Colon/metabolism , Colon/pathology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Gene Silencing , Humans , Mutagens/metabolism , Rectum/metabolism , Rectum/pathologyABSTRACT
RATIONALE: An efficient and reproducible source of genotype-specific human macrophages is essential for study of human macrophage biology and related diseases. OBJECTIVE: To perform integrated functional and transcriptome analyses of human induced pluripotent stem cell-derived macrophages (IPSDMs) and their isogenic human peripheral blood mononuclear cell-derived macrophage (HMDM) counterparts and assess the application of IPSDM in modeling macrophage polarization and Mendelian disease. METHODS AND RESULTS: We developed an efficient protocol for differentiation of IPSDM, which expressed macrophage-specific markers and took up modified lipoproteins in a similar manner to HMDM. Like HMDM, IPSDM revealed reduction in phagocytosis, increase in cholesterol efflux capacity and characteristic secretion of inflammatory cytokines in response to M1 (lipopolysaccharide+interferon-γ) activation. RNA-Seq revealed that nonpolarized (M0) as well as M1 or M2 (interleukin-4) polarized IPSDM shared transcriptomic profiles with their isogenic HMDM counterparts while also revealing novel markers of macrophage polarization. Relative to IPSDM and HMDM of control individuals, patterns of defective cholesterol efflux to apolipoprotein A-I and high-density lipoprotein-3 were qualitatively and quantitatively similar in IPSDM and HMDM of patients with Tangier disease, an autosomal recessive disorder because of mutations in ATP-binding cassette transporter AI. Tangier disease-IPSDM also revealed novel defects of enhanced proinflammatory response to lipopolysaccharide stimulus. CONCLUSIONS: Our protocol-derived IPSDM are comparable with HMDM at phenotypic, functional, and transcriptomic levels. Tangier disease-IPSDM recapitulated hallmark features observed in HMDM and revealed novel inflammatory phenotypes. IPSDMs provide a powerful tool for study of macrophage-specific function in human genetic disorders as well as molecular studies of human macrophage activation and polarization.
Subject(s)
Cell Culture Techniques , Induced Pluripotent Stem Cells/cytology , Macrophages/metabolism , Tangier Disease/pathology , Transcriptome , ATP Binding Cassette Transporter 1/deficiency , ATP Binding Cassette Transporter 1/genetics , ATP Binding Cassette Transporter 1/physiology , Adult , Aged , Animals , Antigens, Differentiation/analysis , Base Sequence , Cell Differentiation , Cells, Cultured , Cholesterol/metabolism , Embryoid Bodies/cytology , Female , Genotype , Humans , Induced Pluripotent Stem Cells/metabolism , Inflammation , Interferon-gamma/pharmacology , Lipopolysaccharides/pharmacology , Macrophage Activation/drug effects , Macrophages/cytology , Macrophages/drug effects , Male , Mice , Mice, Knockout , Molecular Sequence Data , Phagocytosis , Phenotype , RNA, Messenger/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Tangier Disease/genetics , Tangier Disease/metabolism , Young AdultABSTRACT
Recombinant adeno-associated virus (rAAV)-based gene therapies are expanding in their application. Despite progress in manufacturing, current analytical methods for product quantification and characterization remain largely unchanged. Although critical for product and process development, in-process testing, and batch release, current analytical methods are labor-intensive, costly, and hampered by extended turnaround times and low throughput. The field requires more efficient, cost-effective analytical techniques capable of handling large sample quantities to accelerate product and process development. Here, we evaluated Stunner from Unchained Labs for quantifying and characterizing rAAVs and compared it with established analytical methods. Stunner is a combinatorial analytic technology platform that interpolates ultraviolet-visible (UV-Vis) absorption with static and dynamic light scattering (SLS/DLS) analysis to determine capsid and genomic titer, empty and full capsid ratio, and assess vector size and polydispersity. The platform offers empirical measurements with minimal sample requirements. Upon testing hundreds of rAAV vectors, comprising various serotypes and transgenes, the data show a strong correlation with established analytical methods and exhibit high reproducibility and repeatability. Some analyses can be applied to in-process samples from different purification stages and processes, fulfilling the demand for rapid, high-throughput analysis during development. In sum, the pipeline presented streamlines small- and large-batch analytics.
ABSTRACT
AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50 ng/ml) and insulin (10 nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.
Subject(s)
Aldehyde Reductase/metabolism , Carcinoma, Hepatocellular/metabolism , Epidermal Growth Factor/pharmacology , Liver Neoplasms/metabolism , Transcription Factor AP-1/metabolism , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Animals , Base Sequence , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , DNA Primers/genetics , Female , Genes, fos , Hep G2 Cells , Humans , Insulin/pharmacology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Mice, Nude , Promoter Regions, Genetic , RNA, Small Interfering/genetics , Signal Transduction/drug effects , Transcription Factor AP-1/antagonists & inhibitors , Transcription Factor AP-1/genetics , Up-Regulation/drug effectsABSTRACT
Aldo-keto reductase family 1 member B10 (AKR1B10) is primarily expressed in the normal human colon and small intestine but overexpressed in liver and lung cancer. Our previous studies have shown that AKR1B10 mediates the ubiquitin-dependent degradation of acetyl-CoA carboxylase-alpha. In this study, we demonstrate that AKR1B10 is critical to cell survival. In human colon carcinoma cells (HCT-8) and lung carcinoma cells (NCI-H460), small-interfering RNA-induced AKR1B10 silencing resulted in caspase-3-mediated apoptosis. In these cells, the total and subspecies of cellular lipids, particularly of phospholipids, were decreased by more than 50%, concomitant with 2-3-fold increase in reactive oxygen species, mitochondrial cytochrome c efflux, and caspase-3 cleavage. AKR1B10 silencing also increased the levels of alpha,beta-unsaturated carbonyls, leading to the 2-3-fold increase of cellular lipid peroxides. Supplementing the HCT-8 cells with palmitic acid (80 mum), the end product of fatty acid synthesis, partially rescued the apoptosis induced by AKR1B10 silencing, whereas exposing the HCT-8 cells to epalrestat, an AKR1B10 inhibitor, led to more than 2-fold elevation of the intracellular lipid peroxides, resulting in apoptosis. These data suggest that AKR1B10 affects cell survival through modulating lipid synthesis, mitochondrial function, and oxidative status, as well as carbonyl levels, being an important cell survival protein.
Subject(s)
Aldehyde Reductase/metabolism , Aldehydes/metabolism , Lipids/biosynthesis , Malondialdehyde/metabolism , Aldehyde Reductase/antagonists & inhibitors , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , Cell Survival , Cytochromes c/metabolism , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Lipid Peroxides/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/physiology , Oxidative Stress/drug effects , Palmitic Acid/pharmacology , RNA Interference , Reactive Oxygen Species/metabolism , Rhodanine/analogs & derivatives , Rhodanine/pharmacology , Thiazolidines/pharmacologyABSTRACT
Alpha, beta-unsaturated carbonyls are highly reactive mutagens and carcinogens to which humans are exposed on a daily basis. This study demonstrates that aldo-keto reductase family 1 member B10 (AKR1B10) is a critical protein in detoxifying dietary and lipid-derived unsaturated carbonyls. Purified AKR1B10 recombinant protein efficiently catalyzed the reduction to less toxic alcohol forms of crotonaldehyde at 0.90 microM, 4-hydroxynonenal (HNE) at 0.10 microM, trans-2-hexanal at 0.10 microM, and trans-2,4-hexadienal at 0.05 microM, the concentrations at or lower than physiological exposures. Ectopically expressed AKR1B10 in 293T cells eliminated immediately HNE at 1 (subtoxic) or 5 microM (toxic) by converting to 1,4-dihydroxynonene, protecting the cells from HNE toxicity. AKR1B10 protein also showed strong enzymatic activity toward glutathione-conjugated carbonyls. Taken together, our study results suggest that AKR1B10 specifically expressed in the intestine is physiologically important in protecting the host cell against dietary and lipid-derived cytotoxic carbonyls.
Subject(s)
Aldehyde Reductase/metabolism , Carcinogens/metabolism , Cytotoxins/metabolism , Intestines/enzymology , Mutagens/metabolism , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Cell Line , Diet , Humans , Inactivation, Metabolic , Intestinal Mucosa/metabolism , Lipid MetabolismABSTRACT
BACKGROUND: Cyclin B1, the regulatory subunit of cyclin-dependent kinase 1 (Cdk1), is essential for the transition from G2 phase to mitosis. Cyclin B1 is very often found to be overexpressed in primary breast and cervical cancer cells as well as in cancer cell lines. Its expression is correlated with the malignancy of gynecological cancers. METHODS: In order to explore cyclin B1 as a potential target for gynecological cancer therapy, we studied the effect of small interfering RNA (siRNA) on different gynecological cancer cell lines by monitoring their proliferation rate, cell cycle profile, protein expression and activity, apoptosis induction and colony formation. Tumor formation in vivo was examined using mouse xenograft models. RESULTS: Downregulation of cyclin B1 inhibited proliferation of several breast and cervical cancer cell lines including MCF-7, BT-474, SK-BR-3, MDA-MB-231 and HeLa. After combining cyclin B1 siRNA with taxol, we observed an increased apoptotic rate accompanied by an enhanced antiproliferative effect in breast cancer cells. Furthermore, control HeLa cells were progressively growing, whereas the tumor growth of HeLa cells pre-treated with cyclin B1 siRNA was strongly inhibited in nude mice, indicating that cyclin B1 is indispensable for tumor growth in vivo. CONCLUSION: Our data support the notion of cyclin B1 being essential for survival and proliferation of gynecological cancer cells. Concordantly, knockdown of cyclin B1 inhibits proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/drug therapy , Cyclin B/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Paclitaxel/therapeutic use , RNA, Small Interfering/therapeutic use , Uterine Cervical Neoplasms/drug therapy , Animals , Apoptosis , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin B/genetics , Cyclin B/metabolism , Cyclin B1 , Down-Regulation , Female , HeLa Cells , Humans , Mice , Mice, Nude , Neoplasm Proteins/metabolism , Transfection/methods , Tumor Stem Cell Assay , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathologyABSTRACT
Aldo-keto reductase family 1 B10 (AKR1B10), a member of aldo-keto reductase superfamily, is overexpressed in human hepatocellular carcinoma, lung squamous cell carcinoma and lung adenocarcinoma. Our previous study had demonstrated that the ectopic expression of AKR1B10 in 293T cells promotes cell proliferation. To evaluate its potential as a target for cancer intervention, in the current study we knocked down AKR1B10 expression in HCT-8 cells derived from a colorectal carcinoma, using chemically synthesized small interfering RNA (siRNA). The siRNA 1, targeted to encoding region, downregulated AKR1B10 expression by more than 60%, and siRNA 2, targeted to 3' untranslational region, reduced AKR1B10 expression by more than 95%. AKR1B10 silencing resulted in approximately a 50% decrease in cell growth rate and nearly 40% suppression of DNA synthesis. More importantly, AKR1B10 downregulation significantly reduced focus formation rate and colony size in semisolid culture, indicating the critical role of AKR1B10 in HCT-8 cell proliferation. Recombinant AKR1B10 protein showed strong enzymatic activity to acrolein and crotonaldehyde, with K(m) = 110.1 +/- 12.2 microM and V(max) = 3,122.0 +/- 64.7 nmol/mg protein/min for acrolein and K(m) = 86.7 +/- 14.3 microM and V(max) = 2,647.5 +/- 132.2 nmol/mg protein/min for crotonaldehyde. AKR1B10 downregulation enhanced the susceptibility of HCT-8 cells to acrolein (25 microM) and crotonaldehyde (50 microM), resulting in rapid oncotic cell death characterized with lactate dehydrogenase efflux and annexin-V staining. These results suggest that AKR1B10 may regulate cell proliferation and cellular response to additional carbonyl stress, thus being a potential target for cancer intervention.
Subject(s)
Aldehyde Reductase/genetics , Aldehyde Reductase/metabolism , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/pathology , Gene Silencing , Acrolein/toxicity , Aldehydes/toxicity , Aldo-Keto Reductases , Cell Line, Tumor , Cell Proliferation/drug effects , Colorectal Neoplasms/genetics , DNA/biosynthesis , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Kinetics , RNA, Small Interfering/genetics , Sensitivity and SpecificityABSTRACT
Acrolein is a highly reactive alpha,beta-unsaturated aldehyde produced endogenously during lipid peroxidation and naturally distributed pervasively in living environments, posing serious threats to human health if not properly metabolized. In this study, we report aldose reductase-like-1 (ARL-1) as a novel enzyme that catalyzes the reduction of acrolein and protects cells from their toxicity. Using purified ARL-1 protein, we determined its enzymatic activity in response to acrolein and defined its steady-state kinetics with K(m) and V(max) at 0.110 +/- 0.012 mM and 3122.0 +/- 64.7 nmol/mg protein/min, respectively. By introducing a functional Enhanced Green Fluorescent Protein (EGFP)/ARL-1 fusion protein into 293T cells, we demonstrated that plating efficiency in liquid culture and focus formation in soft agar increased by more than 60% (p < 0.05), compared to the vector control cells. More significantly, at a low dose of 5 microM acrolein, EGFP/ARL-1 expression enhanced both plating efficiency and focus formation by more than threefold, and the foci (in soft agar) of 293T cells expressing EGFP/ARL-1 were significantly larger than those of the vector control cells. At high concentrations of acrolein (25 and 50 microM), EGFP/ARL-1 protein prevented oncotic death of 293T cells induced by acrolein. In summary, our data demonstrated for the first time that the ARL-1 protein protects 293T cells from acrolein toxicity. Due to the high toxicity and wide distribution of acrolein, this finding is important to the understanding of its detoxification mechanisms.
Subject(s)
Acrolein/toxicity , Aldehyde Reductase/biosynthesis , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Blotting, Western , Cell Line, Tumor , Cells, Cultured , Colony-Forming Units Assay , Culture Media , DNA Fragmentation/drug effects , Green Fluorescent Proteins/biosynthesis , Green Fluorescent Proteins/genetics , Humans , Kinetics , L-Lactate Dehydrogenase/metabolism , Recombinant Proteins/metabolism , TransfectionABSTRACT
Despite the recognized role of the ATP-binding Cassette Transporter A1 (ABCA1) in high-density lipoprotein (HDL) metabolism, our understanding of ABCA1 deficiency in human hepatocytes is limited. To define the functional effects of human hepatocyte ABCA1 deficiency, we generated induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) from Tangier disease (TD) and matched control subjects. Control HLCs exhibited robust cholesterol efflux to apolipoprotein A-I (apoA-I) and formed nascent HDL particles. ABCA1-deficient HLCs failed to mediate lipid efflux or nascent HDL formation, but had elevated triglyceride (TG) secretion. Global transcriptome analysis revealed significantly increased ANGPTL3 expression in ABCA1-deficient HLCs. Angiopoietin-related protein 3 (ANGPTL3) was enriched in plasma of TD relative to control subjects. These results highlight the required role of ABCA1 in cholesterol efflux and nascent HDL formation by hepatocytes. Furthermore, our results suggest that hepatic ABCA1 deficiency results in increased hepatic TG and ANGPTL3 secretion, potentially underlying the elevated plasma TG levels in TD patients.
Subject(s)
ATP Binding Cassette Transporter 1/metabolism , Lipoproteins, HDL/metabolism , ATP Binding Cassette Transporter 1/genetics , Angiopoietin-Like Protein 3 , Angiopoietin-like Proteins/blood , Angiopoietin-like Proteins/genetics , Angiopoietin-like Proteins/metabolism , Apolipoprotein A-I/metabolism , Cell Differentiation , Cells, Cultured , Cholesterol/metabolism , Hepatocytes/cytology , Hepatocytes/metabolism , Humans , Induced Pluripotent Stem Cells/cytology , Tangier Disease/metabolism , Tangier Disease/pathology , Transcriptome , Triglycerides/metabolismABSTRACT
Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.
Subject(s)
Genetic Loci , Genetic Variation , Hepatocytes/cytology , Induced Pluripotent Stem Cells/cytology , Lipids/blood , Animals , Base Sequence , Cohort Studies , Gene Expression Profiling , Genetic Predisposition to Disease , Genome-Wide Association Study , Hepatocytes/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Lipid Metabolism/genetics , Liver/metabolism , Mice , Organ Specificity/genetics , Polymorphism, Single Nucleotide/genetics , Quantitative Trait Loci/geneticsABSTRACT
Uridine phosphorylase (UPase) is an enzyme that converts the pyrimidine nucleoside uridine into uracil. Upon availability of ribose-1-phosphate, UPase can also catalyze the formation of nucleosides from uracil as well as from 5-fluorouracil, therefore involved in fluoropyrimidine metabolism. UPase gene expression is strictly controlled at the promoter level by oncogenes, tumor suppressor genes, and cytokines. UPase activity is usually elevated in various tumor tissues, including breast cancer, compared to matched normal tissues and this induction appears to contribute to the therapeutic efficacy of fluoropyrimidines in cancer patients. In this review, we will discuss in detail the role of UPase in the activation of fluoropyrimidines and its effect on the prognosis of breast cancer patients.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/enzymology , Uridine Phosphorylase/biosynthesis , Uridine Phosphorylase/metabolism , Animals , Antimetabolites, Antineoplastic/metabolism , Antimetabolites, Antineoplastic/therapeutic use , Biomarkers, Tumor , Capecitabine , Deoxycytidine/analogs & derivatives , Deoxycytidine/metabolism , Enzyme Induction , Female , Floxuridine/metabolism , Floxuridine/therapeutic use , Fluorouracil/metabolism , Fluorouracil/therapeutic use , Gene Expression Regulation , Humans , Mice , Prodrugs/metabolism , Prognosis , Pyrimidinones , Treatment OutcomeABSTRACT
Cyclin B1 is the regulatory subunit of M-phase promoting factor, and proper regulation of cyclin B1 is essential for the initiation of mitosis. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting the suppression of cyclin B1 could be an attractive strategy for antiproliferative therapy. In the present work, we analysed the impact of small interfering RNAs (siRNAs) targeted to cyclin B1 on different human tumor cell lines. Cyclin B1 siRNAs reduced the protein level of cyclin B1 in HeLa, MCF-7, BT-474 and MDA-MB-435 tumor cells and efficiently reduced the kinase activity of Cdc2/cyclin B1 in HeLa cells. siRNA-treated cells were arrested in G2/M phase in all tumor cell lines tested. Proliferation of tumor cells from different origins was suppressed by 50-80% 48 h after transfection and apoptosis was increased from 5 to 40-50%. Furthermore, tumor cells showed less colony-forming ability after siRNA treatment. In contrast, primary human umbilical vein endothelial cells exhibited only a slight change in cell cycle, and neither apoptosis nor clear inhibition of proliferation was observed after cyclin B1 siRNA treatment for 48 h. These results indicate that siRNAs against cyclin B1 could become a powerful antiproliferative tool in future antitumor therapy.
Subject(s)
Apoptosis/genetics , Cyclin B/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , CDC2 Protein Kinase/drug effects , CDC2 Protein Kinase/metabolism , Cell Division/genetics , Cells, Cultured , Cyclin B/drug effects , Cyclin B/metabolism , Cyclin B1 , Down-Regulation , Female , G2 Phase/drug effects , G2 Phase/genetics , HeLa Cells , Humans , Maturation-Promoting Factor/drug effects , Maturation-Promoting Factor/metabolism , Mitosis/drug effects , Mitosis/genetics , Neoplastic Stem Cells , RNA, Small Interfering/pharmacology , Tumor Cells, Cultured , Umbilical Veins/cytologyABSTRACT
Mitochondria are dynamic organelles that undergo constant fission and fusion cycles. In response to cellular damage, this balance is shifted dramatically toward fission. Cyclin C-Cdk8 kinase regulates transcription of diverse gene sets. Using knockout mouse embryonic fibroblasts (MEFs), we demonstrate that cyclin C directs the extensive mitochondrial scission induced by the anticancer drug cisplatin or oxidative stress. This activity is independent of transcriptional regulation, as Cdk8 is not required for this activity. Furthermore, adding purified cyclin C to unstressed permeabilized MEF cultures induced complete mitochondrial fragmentation that was dependent on the fission factors Drp1 and Mff. To regulate fission, a portion of cyclin C translocates from the nucleus to the cytoplasm, where it associates with Drp1 and is required for its enhanced mitochondrial activity in oxidatively stressed cells. In addition, although HeLa cells regulate cyclin C in a manner similar to MEF cells, U2OS osteosarcoma cultures display constitutively cytoplasmic cyclin C and semifragmented mitochondria. Finally, cyclin C, but not Cdk8, is required for loss of mitochondrial outer membrane permeability and apoptosis in cells treated with cisplatin. In conclusion, this study suggests that cyclin C connects stress-induced mitochondrial hyperfission and programmed cell death in mammalian cells.
Subject(s)
Apoptosis , Cyclin C/physiology , Mitochondrial Dynamics , Animals , Dynamins/metabolism , HeLa Cells , Humans , Mice , Mitochondria/metabolism , Protein Transport , Signal Transduction , Stress, PhysiologicalABSTRACT
PURPOSE: Ulcerative colitis and colitis-associated colorectal cancer (CAC) is a serious health issue, but etiopathological factors remain unclear. Aldo-keto reductase 1B10 (AKR1B10) is specifically expressed in the colonic epithelium, but downregulated in colorectal cancer. This study was aimed to investigate the etiopathogenic role of AKR1B10 in ulcerative colitis and CAC. EXPERIMENTAL DESIGN: Ulcerative colitis and CAC biopsies (paraffin-embedded sections) and frozen tissues were collected to examine AKR1B10 expression. Aldo-keto reductase 1B8 (the ortholog of human AKR1B10) knockout (AKR1B8(-/-)) mice were produced to estimate its role in the susceptibility and severity of chronic colitis and associated dysplastic lesions, induced by dextran sulfate sodium (DSS) at a low dose (2%). Genome-wide exome sequencing was used to profile DNA damage in DSS-induced colitis and tumors. RESULTS: AKR1B10 expression was markedly diminished in over 90% of ulcerative colitis and CAC tissues. AKR1B8 deficiency led to reduced lipid synthesis from butyrate and diminished proliferation of colonic epithelial cells. The DSS-treated AKR1B8(-/-) mice demonstrated impaired injury repair of colonic epithelium and more severe bleeding, inflammation, and ulceration. These AKR1B8(-/-) mice had more severe oxidative stress and DNA damage, and dysplasias were more frequent and at a higher grade in the AKR1B8(-/-) mice than in wild-type mice. Palpable masses were seen in the AKR1B8(-/-) mice only, not in wild-type. CONCLUSIONS: AKR1B8 is a critical protein in the proliferation and injury repair of the colonic epithelium and in the pathogenesis of ulcerative colitis and CAC, being a new etiopathogenic factor of these diseases.
Subject(s)
Alcohol Oxidoreductases/genetics , Colitis, Ulcerative/pathology , Colon/pathology , Intestinal Mucosa/pathology , Oxidoreductases Acting on Aldehyde or Oxo Group Donors/metabolism , Alcohol Oxidoreductases/biosynthesis , Alcohol Oxidoreductases/metabolism , Aldo-Keto Reductases , Animals , Base Sequence , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Colitis, Ulcerative/chemically induced , Colorectal Neoplasms/pathology , DNA Damage/genetics , Dextran Sulfate , Disease Models, Animal , Epithelial Cells/metabolism , Humans , Inflammation/genetics , Inflammation/immunology , Inflammation/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress/genetics , Oxidoreductases Acting on Aldehyde or Oxo Group Donors/biosynthesis , Oxidoreductases Acting on Aldehyde or Oxo Group Donors/genetics , Reactive Oxygen Species/metabolism , Sequence Analysis, DNAABSTRACT
OBJECTIVE: We propose to block the autocrine and (or) paracrine pathway of vascular endothelial growth factor (VEGF) in ovarian cancer by using anti-VEGF hairpin ribozyme gene to see whether the growth of tumor cells could be inhibited and to further exploit its mechanisms. METHODS: Anti-VEGF hairpin ribozyme gene eukaryotic expression vector was introduced into ovarian cancer cells SKOV(3) by using lipofectin mediation and positive clones were screened by genticin (G418); ribozyme (RZ) expression was confirmed by RNA dot blot; the VEGF expression of SKOV(3) cells before or after transfection were detected by reverse transcription polymerase chain reaction (RT-PCR) method, transmission electron microscopy demonstrated the morphologic changes of ribozyme-generating SKOV(3) cells; the growth of transfected cells in nude mice were detected. RESULTS: VEGF expression was decreased distinctly in SKOV(3) cells transfected by RZ (SKOV(3)-RZ) cells. Apoptosis cells were observed; the formation rate and growth speed of xenografted tumor slowed down. CONCLUSIONS: Anti-VEGF hairpin ribozyme gene can inhibit the expression of VEGF mRNA and VEGF in SKOV(3) cells. The growth of xenografted tumor in nude mice was inhibited by reduced angiogenesis. This provides an experimental basis for curing human ovarian cancer with antiangiogenesis method.
Subject(s)
Endothelial Growth Factors/antagonists & inhibitors , Lymphokines/antagonists & inhibitors , Ovarian Neoplasms/therapy , RNA, Catalytic/therapeutic use , Animals , Endothelial Growth Factors/genetics , Endothelial Growth Factors/physiology , Female , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/physiology , Lymphokines/genetics , Lymphokines/physiology , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Neoplasm Transplantation , Ovarian Neoplasms/pathology , Transfection , Transplantation, Heterologous , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
Abrogation of uridine phosphorylase (UPase) leads to abnormalities in pyrimidine metabolism and host protection against 5-fluorouracil (5-FU) toxicity. We elucidated the effects on the metabolism and antitumor efficacy of 5-FU and capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) in our UPase knockout (UPase(-/-)) model. Treatment with 5-FU (85 mg/kg) or capecitabine (1,000 mg/kg) five days a week for four weeks caused severe toxicity and structural damage to the intestines of wild-type (WT) mice, but not in UPase(-/-) animals. Capecitabine treatment resulted in a 70% decrease in blood cell counts of WT animals, with only a marginal effect in UPase(-/-) mice. UPase expressing colon 38 tumors implanted in UPase(-/-) mice revealed an improved therapeutic efficacy when treated with 5-FU and capecitabine because of the higher maximum tolerated dose for fluoropyrimidines achievable in UPase(-/-) mice. (19)F-MRS evaluation of capecitabine metabolism in tumors revealed similar activation of the prodrug in UPase(-/-) mice compared with WT. In WT mice, approximately 60% of capecitabine was transformed over three hours into its active metabolites, whereas 80% was transformed in tumors implanted in UPase(-/-) mice. In UPase(-/-) mice, prolonged retention of 5'dFUR allowed a proportional increase in tumor tissue. The similar presence of fluorinated catabolic species confirms that dihydropyrimidine dehydrogenase activity was not altered in UPase(-/-) mice. Overall, these results indicate the importance of UPase in the activation of fluoropyrimidines, the effect of uridine in protecting normal tissues, and the role for tumor-specific modulation of the phosphorolytic activity in 5-FU or capecitabine-based chemotherapy.
Subject(s)
Fluorouracil/therapeutic use , Neoplasms/drug therapy , Neoplasms/genetics , Uridine Phosphorylase/genetics , Animals , Antimetabolites, Antineoplastic/metabolism , Antimetabolites, Antineoplastic/therapeutic use , Capecitabine , Cell Line, Tumor , Deoxycytidine/analogs & derivatives , Deoxycytidine/therapeutic use , Drug Evaluation, Preclinical , Drug Resistance, Neoplasm/genetics , Fluorouracil/analogs & derivatives , Fluorouracil/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Gene Expression Regulation, Enzymologic/physiology , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/enzymology , Neoplasms/metabolism , Prodrugs/metabolism , Prodrugs/therapeutic use , Pyrimidines/metabolism , Pyrimidines/therapeutic use , Treatment Outcome , Uridine Phosphorylase/metabolism , Uridine Phosphorylase/physiologyABSTRACT
Recent studies have demonstrated that aldo-keto reductase family 1 B10 (AKR1B10), a novel protein overexpressed in human hepatocellular carcinoma and non-small cell lung carcinoma, may facilitate cancer cell growth by detoxifying intracellular reactive carbonyls. This study presents a novel function of AKR1B10 in tumorigenic mammary epithelial cells (RAO-3), regulating fatty acid synthesis. In RAO-3 cells, Sephacryl-S 300 gel filtration and DEAE-Sepharose ion exchange chromatography demonstrated that AKR1B10 exists in two distinct forms, monomers (approximately 40 kDa) bound to DEAE-Sepharose column and protein complexes (approximately 300 kDa) remaining in flow-through. Co-immunoprecipitation with AKR1B10 antibody and protein mass spectrometry analysis identified that AKR1B10 associates with acetyl-CoA carboxylase-alpha (ACCA), a rate-limiting enzyme of de novo fatty acid synthesis. This association between AKR1B10 and ACCA proteins was further confirmed by co-immunoprecipitation with ACCA antibody and pulldown assays with recombinant AKR1B10 protein. Intracellular fluorescent studies showed that AKR1B10 and ACCA proteins co-localize in the cytoplasm of RAO-3 cells. More interestingly, small interfering RNA-mediated AKR1B10 knock down increased ACCA degradation through ubiquitination-proteasome pathway and resulted in >50% decrease of fatty acid synthesis in RAO-3 cells. These data suggest that AKR1B10 is a novel regulator of the biosynthesis of fatty acid, an essential component of the cell membrane, in breast cancer cells.
Subject(s)
Acetyl-CoA Carboxylase/chemistry , Aldehyde Reductase/physiology , Breast Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Acetyl-CoA Carboxylase/metabolism , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Cell Line, Tumor , Cell Membrane/metabolism , Cytoplasm/metabolism , Fatty Acids/metabolism , Gene Silencing , Humans , Proteasome Endopeptidase Complex/metabolism , RNA, Small Interfering/metabolism , Recombinant Proteins/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Ubiquitin/metabolismABSTRACT
Uridine phosphorylase (UPase) has been shown to play an important role in the antineoplastic activity of 5-fluorouracil (5-FU) and in the anabolism of its oral prodrug, capecitabine, through the conversion of 5'-deoxy-5-fluorouridine (5'-DFUR) into 5-FU. In this study, we investigated the effect of tumor necrosis factor-alpha (TNF-alpha) on UPase gene expression and 5'-DFUR antiproliferative activity and elucidated the involved signal transduction pathway. Our data indicate that TNF-alpha significantly induced UPase mRNA expression and its enzymatic activity in EMT6 murine breast cancer cells, leading to an enhanced cytotoxicity of 5'-DFUR. This is further confirmed by an increased incorporation of 5'-DFUR-originated 5-FU nucleotides into nucleic acids. To clarify the mechanism of TNF-alpha-induced UPase expression, we first observed the effect of TNF-alpha on the UPase promoter activity with a series of 5'-deleted promoter-luciferase constructs. Transient transfection analysis showed that the TNF-alpha-inductive pattern in EMT6 cells was consistent with the presence of a nuclear factor-kappaB (NF-kappaB) binding element (-1332/-1312 bp) in the UPase promoter region. Furthermore, electrophoretic mobility shift assays, supershift, and cotransfection assays revealed that the activation of p65 was responsible for UPase induction by TNF-alpha. Finally, the induction of UPase by TNF-alpha could be suppressed by PS-341, a NF-kappaB inhibitor. In summary, TNF-alpha efficiently induces UPase gene expression through a NF-kappaB subunit p65-dependent pathway enhancing cell sensitivity to 5'-DFUR. The elucidation of this regulation mechanism may aid in the clinical use of 5-FU-based chemotherapy.